We continue to develop a project designed to to understand the regulation of key virulence factors in Borrelia hermsii that are required for successful transmission from its tick vector, Ornithodoros hermsi, to a mammalian host. While B. hermsii has the genes encoding similar regulatory proteins as B. burgdorferi (i.e., RpoS, RpoD and the Borrelia oxidative stress regulator, BosR), the responses of the bacterium to environmental signals, such as temperature, reactive oxygen species (ROS), reactive nitrogen species (RNS), dissolved oxygen and pH, are dramatically different. These differences reflect a finely tuned adaptation by these pathogenic spirochetes to the physiology of their particular arthropod vectors. A hallmark of gene regulation in B. burgdorferi is the RpoN/RpoS regulatory network. This regulatory cascade coordinates the expression of virulence factors required for the successful transmission of B. burgdorferi. However, all strains of relapsing fever spirochetes isolated in North America do not harbor a functional RpoN, suggesting that the RpoN/RpoS regulatory cascade does not function in B. hermsii. Therefore, our initial focus has been on developing the necessary biochemical and genetic tools to study the RpoS-, RpoD- and BosR-dependent gene regulation of known virulence factors (i.e., Vmp and Vtp) in B. hermsii with a particular emphasis on the regulation of switching from Vtp to Vmp during the transmission cycle. In FY2019, our research group made significant progress improving the genetic tools necessary for studying B. hermsii. Several reports over the past five years have shown that the expression of the Variable tick protein (Vtp) is required for successful transmission of relapsing fever (RF) spirochetes from O. hermsi and that up-regulation of Vtp occurs in the salivary glands of ticks before the initiation of feeding. This suggests that conditions in the resting salivary glands induce the expression of this important protein prior to feeding in order to promote rapid and successful transmission to a new mammalian host. Our preliminary data suggests that ROS is present at significant levels in resting tick salivary glands. Significantly, ROS (e.g., hydrogen peroxide or t-butyl peroxide) can induce the expression of Vtp in vitro. These observations indicate that Vtp expression might be regulated by BosR. To dissect the BosR-dependent regulation of Vtp, we attempted to generate a BosR mutant in B. hermsii but multiple attempts were unsuccessful. Therefore, we adapted a B. burgdorferi LacI inducible gene expression system for use in B. hermsii. In addition, we developed an efficient plating method to allow us to isolate clonal bacteria directly from the blood of infected mice and infected ticks. We have used this method to isolate arginine deiminase (AcrA) mutants and LacI-CsrA (carbon storage regulatory repressor) strains of B. hermsii. The development of these systems and methods are pivotal for analyzing the regulatory networks in B. hermsii that are required for successful transmission and for evaluating the role of arginine and oxygen in promoting rapid growth and high cell density of B. hermsii in the blood of infected animals.